Damage-regulated Autophagy Modulator Expression Research Articles

Different effects of anti-VEGF drugs (Ranibizumab, Aflibercept, Conbercept) on autophagy and its effect on neovascularization in RF/6A cells

IntroductionChoroidal neovascularization (CNV) is the main pathological change of wet age-related macular degeneration. Anti-VEGF drugs are the most commonly used treatment for CNV. The biggest drawback of anti-VEGF drugs is the recurrence of CNV, which requires repeated therapy several times. Autophagy activation may be involved in reducing the therapeutic effect of anti-VEGF drugs. So, this study aims to elucidate the effect and mechanism of anti-VEGF drugs on endothelial autophagy and neovascularization in vitro. MethodsRF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2, 5% CO2, 94% N2), anti-VEGF group (group1: Ranibizumab 100 μg/ml; group2: Aflibercept, 400 μg/ml; group3: Conbercept, 100 μg/ml). Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. Subsequently, we used autophagy inhibitors (3-MA or CQ) to inhibit Conbercept induced autophagy and to observe its effect on angiogenesis in vitro. Proliferation, migration, and tube formation of endothelial cells can be used to study neovascularization in vitro. In this research, the CCK-8 assay was used to detect cell proliferation. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Flow cytometry and Tunel were used to detect cell apoptosis. Finally, the mechanism of Conbercept activated autophagy was studied. Western blot was used to detect the expression of p53 and DRAM (damage-regulated autophagy modulator), upstream activators of autophagy. ResultsThe protein levels of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher than in the hypoxia group(P < 0.05). While the expression of P62 was decreased (P < 0.05). The autophagic flux was showed the same results. However, Aflibercept showed the opposite effect on autophagy. Compared with the Conbercept group, autophagy inhibitor 3-MA or CQ can further inhibit cell proliferation and promotes cell apoptosis (P < 0.05). Conbercept significantly inhibited cell migration compared with the hypoxia group (633.08 ± 72.52 vs. 546.33 ± 24.61), while the autophagy inhibitor group (3-MA or CQ) had a more obvious inhibition effect (309.75 ± 86.36 and 263.33 ± 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in the Conbercept group (32.00 ± 2.00) compared to the hypoxia group (39.00 ± 1.53) and even further reduced in 3-MA or CQ group (24.00 ± 3.61, 20.00 ± 2.65). The length of master segments in the hypoxia group was 15,668.00 ± 894.11. It was decreased in Conbercept (13,885.34 ± 730.03). In 3-MA or CQ group, the length of master segments dropped further (11,997.00 ± 433.66, 10,617.67 ± 543.21). Compare with the hypoxia group, the expression P53 and DRAM were increased in the Conbercept group (P < 0.05). Autophagy-related proteins LC-3, Beclin-1, and DRAM were inhibited by P53 inhibitor Pifithrin-α (PFTα) (P < 0.05). ConclusionRanibizumab and Conbercept can trigger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of Conbercept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of the p53/DRAM pathway.

DRAM is Involved in Hypoxia/Ischemia-Induced Autophagic Apoptosis in Hepatocytes.

Liver hypoxia/ischemia injury leads to acute liver injury, delayed graft dysfunction, and failure during liver transplantation. Previous studies showed that autophagy is involved in liver hypoxia/ischemia injury. Our and others’ studies have found that the damage-regulated autophagy modulator (DRAM) could induce the autophagic apoptosis. However, the role of DRAM regulating autophagy in liver hypoxia/ischemia injury remains unclear. The aim of this study was to determine whether DRAM is involved in oxygen-glucose deprivation (OGD)-induced hepatocyte autophagic apoptosis. Normal hepatocytes (HL-7702) were treated with OGD while Balb/c mice underwent surgery to induce 70% liver ischemia. To evaluate the role of DRAM in hypoxia/ischemia-induced hepatic injury, DRAM siRNA was used to knockdown DRAM expression in cultured hepatocytes and a recombinant adenovirus vector expressing DRAM was used to overexpress DRAM in cultured hepatocytes in vitro and in the liver in vivo. Hepatic injury was analyzed by histopathological methods and measurement of hepatocyte enzyme release. Cell apoptosis was analyzed by flow cytometry and TUNEL staining. Several autophagic biomarkers were observed by western blot analysis. OGD and 70% hepatic ischemia significantly induced cell autophagy, apoptosis and DRAM expression in hepatocytes in vitro and in vivo. OGD-induced autophagic apoptosis was inhibited by 3-Methyladenine (3-MA). OGD-induced injury and autophagy in HL-7702 cells were significantly attenuated by DRAM knockdown but aggravated by DRAM overexpression in vitro. Similarly, DRAM overexpression increased ischemia-induced liver injury and hepatic apoptosis in vivo. Our data demonstrate that hypoxia/ischemia induces hepatic injury through a DRAM-dependent autophagic apoptosis pathway. These data also suggest that DRAM plays an important role in ischemia-induced liver injury and hepatocyte apoptosis.

P53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions

BackgroundAs an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA.Methods and resultsHere we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression.ConclusionsAltogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo.

Anti-cancer effect of Scutellaria baicalensis in combination with cisplatin in human ovarian cancer cell

BackgroundOvarian cancer is one of the major causes of death among females in worldwide. Cisplatin is a primary anti-cancer drug against ovarian cancer, but the recurrent tumors after treatment frequently show acquired chemoresistance. Extract of Scutellaria baicalensis (SbE) has been reported to have functional compounds including baicalin, which has anti-cancer effects. However, the anti-cancer effects of SbE in ovarian cancer and its underlying mechanisms are elusive.MethodsWe investigated that the effects of SbE and/or cisplatin on cell death in the cisplatin sensitive ovarian cancer cell line A2780 (CSC) and the counterpart cell line that has cisplatin resistance (CRC). Molecular mechanisms of the effects, focusing on apoptosis and autophagy, were examined.ResultsTreatment of cisplatin or SbE reduced cell viability significantly in CSC and too much lesser extent in CRC. Cisplatin-induced cell death in CSC was mediated by p53-induced apoptosis acompanied by expresson of damage-regulated autophagy modulator (DRAM). In CRC, decreased DRAM expression (p < 0.01) hindered p21-mediated cell death and contributed to cisplatin resistance. Treatment of SbE also induced cell death in CSC by p53-dependent apoptosis, not in CRC. Autophagy was not induced by neither cisplatin nor SbE. Intriguingly, the combinational treatment of SbE and cisplatin significantly decreased cell viability in CRC. The cell death was mediated by autophagy with increased expression of Atg5 and Atg12 (p < 0.05), rather than p53-dependent pathway with repressed expression of p21 (p < 0.001) through HDAC1 activation.ConclusionsThe combined treatment of SbE with cisplatin was effective in CRC, leading to cell death via Beclin1-independent autophagy, suggesting that SbE treatment in combination with cisplatin has a potential as a chemotherapeutic agent in cisplatin-resistant ovarian cancer.

Psammaplin A induces Sirtuin 1-dependent autophagic cell death in doxorubicin-resistant MCF-7/adr human breast cancer cells and xenografts

BackgroundPsammaplin A (PsA) is a natural product isolated from marine sponges, which has been demonstrated to have anticancer activity against several human cancer cell lines via the induction of cell cycle arrest and apoptosis. New drugs that are less toxic and more effective against multidrug-resistant cancers are urgently needed. MethodsWe tested cell proliferation, cell cycle progression and autophagic cell death pathway in doxorubicin-resistant MCF-7 (MCF-7/adr) human breast cancer cells. The potency of PsA was further determined using an in vivo xenograft model. Results and conclusionPsA significantly inhibited MCF-7/adr cells proliferation in a concentration-dependent manner, with accumulation of cells in G2/M phase of the cell cycle. PsA significantly decreased SIRT1 enzyme activity and reduced expression of SIRT1 protein in the cultured cells with greater potency than sirtinol or salermide. Acetylation of p53, a putative target of SIRT1, increased significantly following PsA treatment. In addition, PsA markedly increased the expression levels of autophagy-related proteins. In support of this, it was found that PsA significantly increased the expression of damage-regulated autophagy modulator (DRAM), a p53-induced protein. General significanceThe results of this study suggest that PsA is sufficient to overcome multidrug-resistant cancer via SIRT1-mediated autophagy in MCF-7/adr breast cancer cells, indicating that PsA has therapeutic potential for clinical use.

CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2.

Apoptosis-stimulating protein of p53-2 (ASPP2) induces apoptosis by promoting the expression of pro-apoptotic genes via binding to p53 or p73; however, the exact mechanisms by which ASPP2 induces apoptotic death in hepatoma cells are still unclear. Here, we show that the transient overexpression of ASPP2 induces autophagic apoptosis in hepatoma cells by promoting p53- or p73-independent C/EBP homologous protein (CHOP) expression. CHOP expression decreases the expression of Bcl-2; this change releases Beclin-1 from cytoplasmic Bcl-2-Beclin-1 complexes and allows it to initiate autophagy. However, transient overexpression of Beclin-1 can induce autophagy but not apoptosis. Our results show that ASPP2 induces the expression of damage-regulated autophagy modulator (DRAM), another critical factor that cooperates with free Beclin-1 to induce autophagic apoptosis. The effect of CHOP on the translocation and sequestration of Bcl-2 in the nucleus, which requires the binding of Bcl-2 to ASPP2, is also critical for ASPP2-induced autophagic apoptosis. Although the role of nuclear ASPP2–Bcl-2 complexes is still unclear, our results suggest that nuclear ASPP2 can prevent the translocation of the remaining Bcl-2 to the cytoplasm by binding to Bcl-2 in a CHOP-dependent manner, and this effect also contributes to Beclin-1-initiated autophagy. Thus, CHOP is critical for mediating ASPP2-induced autophagic apoptosis by decreasing Bcl-2 expression and maintaining nuclear ASPP2–Bcl-2 complexes. Our results, which define a mechanism whereby ASPP2 overexpression induces autophagic apoptosis, open a new avenue for promoting autophagy in treatments to cure hepatocellular carcinoma.

Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria

Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma.

Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM‐induced autophagy or BAX

Background & AimsApoptosis mediated by p53 plays a pathological role in the progression of hepatosteatosis. It is noteworthy that p53 can promote the expression of damage-regulated autophagy modulator (DRAM), an inducer of autophagy-mediated apoptosis. However, the relationship between p53-mediated apoptosis and autophagy in hepatosteatosis remains elusive. This study aimed to examine how p53 orchestrates autophagy and apoptosis to affect hepatosteatosis.MethodsHepG2 cells were treated with oleic acid (OA) for 24 h to induce hepatosteatosis. Mice were fed a high-fat diet for 20 or 40 weeks to induce hepatosteatosis.ResultsOA induced a dose-dependent increase in steatosis severity and apoptosis. OA also induced autophagy, which was a critical inducer of apoptosis in mild steatosis induced by 400 μM OA, but not in the more severe steatosis induced by 800 and 1200 μM OA. p53 inhibition by siRNA mostly blocked OA-induced apoptosis and autophagy. Moreover, OA-induced autophagy was DRAM-dependent and primarily occurred in the mitochondria (mitophagy), where DRAM was localized. In severe steatosis induced by 1200 μM OA, apoptosis was mainly dependent on p53-induced expression of BAX, which was also localized to the mitochondria. Our in vivo study showed that p53 expression increased in both mild and severe hepatosteatosis. Increased DRAM expression and autophagy were identified in mild hepatosteatosis, whereas greater BAX expression was observed in severe hepatosteatosis.Conclusionsp53 may induce apoptosis via different mechanisms. DRAM-mediated mitophagy is a primary apoptotic inducer in mild hepatosteatosis, whereas p53-induced BAX expression mainly induces apoptosis in severe hepatosteatosis.

SIRT1: Identification of a novel placental stress-response protein that regulates fetal growth?

Psammaplin A (PsA) is a natural product isolated from marine sponges, which has been demonstrated to have anticancer activity against several human cancer cell lines via the induction of cell cycle arrest and apoptosis. New drugs that are less toxic and more effective against multidrug-resistant cancers are urgently needed.We tested cell proliferation, cell cycle progression and autophagic cell death pathway in doxorubicin-resistant MCF-7 (MCF-7/adr) human breast cancer cells. The potency of PsA was further determined using an in vivo xenograft model.PsA significantly inhibited MCF-7/adr cells proliferation in a concentration-dependent manner, with accumulation of cells in G2/M phase of the cell cycle. PsA significantly decreased SIRT1 enzyme activity and reduced expression of SIRT1 protein in the cultured cells with greater potency than sirtinol or salermide. Acetylation of p53, a putative target of SIRT1, increased significantly following PsA treatment. In addition, PsA markedly increased the expression levels of autophagy-related proteins. In support of this, it was found that PsA significantly increased the expression of damage-regulated autophagy modulator (DRAM), a p53-induced protein.The results of this study suggest that PsA is sufficient to overcome multidrug-resistant cancer via SIRT1-mediated autophagy in MCF-7/adr breast cancer cells, indicating that PsA has therapeutic potential for clinical use.

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.

Mevalonate Cascade Regulation of Airway Mesenchymal Cell Autophagy and Apoptosis: A Dual Role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.

C-Jun NH2-Terminal Kinase Activation Is Essential for DRAM-Dependent Induction of Autophagy and Apoptosis in 2-Methoxyestradiol–Treated Ewing Sarcoma Cells

Ewing sarcoma and osteosarcoma are two aggressive cancers that affect bones and soft tissues in children and adolescents. Despite multimodal therapy, patients with metastatic sarcoma have a poor prognosis, emphasizing a need for more effective treatment. We have shown previously that 2-methoxyestradiol (2-ME), an antitumoral compound, induces apoptosis in Ewing sarcoma cells through c-Jun NH(2)-terminal kinase (JNK) activation. In the present study, we provide evidence that 2-ME elicits macroautophagy, a process that participates in apoptotic responses, in a JNK-dependent manner, in Ewing sarcoma and osteosarcoma cells. We also found that the enhanced activation of JNK by 2-ME is partially regulated by p53, highlighting the relationship of JNK and autophagy to p53 signaling pathway. Furthermore, we showed that 2-ME up-regulates damage-regulated autophagy modulator (DRAM), a p53 target gene, in Ewing sarcoma cells through a mechanism that involves JNK activation. The silencing of DRAM expression reduced both apoptosis and autophagy triggered by 2-ME in Ewing sarcoma and osteosarcoma cells. Our results therefore identify JNK as a novel mediator of DRAM regulation. These findings suggest that 2-ME or other anticancer therapies that increase DRAM expression or function could be used to effectively treat sarcoma patients.